Systems and methods for culturing epithelial cells

ABSTRACT

The present invention features assays for co-culturing primary cells while maintaining key biological activities specific to the primary cells. The invention is based, at least in part, on the discovery that compositions and methods for primary cells in a high-throughput co-culture platform, image analysis for distinguishing cells in co-cultures and assays that are suitable for screening of agents in epithelial cells, such as hepatocytes.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national phase application, pursuant to 35U.S.C. § 371, of PCT international application Ser. No.:PCT/US2014/028219, filed Mar. 14, 2014, designating the United Statesand published in English, which claims the benefit of the following U.S.Provisional Application No. 61/791,798, filed Mar. 15, 2013, entitled“Systems and Methods for Culturing Epithelial Cells,” the entirecontents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with government support under Grant Nos.HG005032, DK065152, DK56966, and GM089652 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Chronic liver disease affects more than 500 million people worldwide.The lack of efficacious treatment options for liver disease is acritical unmet medical need. Most treatments for liver disease arepalliative. The only therapy shown to directly alter outcome and preventmortality is organ transplantation, but its utility is limited by apersistent shortage of donor organs and potential complications arisingfrom host-graft rejection. The deficit of treatment options is furthercompounded by the absence of a predictive in vitro hepatocyte model, acritical tool for advancing the discovery and development of new drugsto treat liver disease.

The universal utility of a predictive in vitro hepatocyte model isshared across drug development efforts and not limited to drugdevelopment for liver disease. A third of drug withdrawals from themarket and more than half of all warning labels on drugs approved for avariety of indications are primarily due to adverse affects on theliver. Moreover, the majority of new drug candidates fail in Phase Iclinical trials due to issues with liver toxicity and bioavailability ofdrug candidates indicating that current in vitro liver models used bythe pharmaceutical industry, though useful in a limited capacity, arenot fully predictive of in vivo liver metabolism and toxicity.

Historically cell culture techniques have failed to take into accountthe necessary microenvironment for cell-cell and cell-matrixcommunication. Hepatocytes in particular are notoriously difficult tomaintain in culture as they rapidly lose viability and phenotypicfunctions. Moreover, the typically complex and multi-layered culturesystems that work effectively are hard to replicate in miniature, as isneeded for preparing such cultures in multi-well plate formats forhigh-throughput screening. While some progress has been made inculturing isolated primary human hepatocytes, adapting these in vitroliver models for high-throughput screening of drugs for theirpharmacological and toxicology effects on hepatocytes has remainedelusive.

SUMMARY OF THE INVENTION

The present invention features assays for co-culturing primary cellswhile maintaining key biological activities specific to the primarycells. The invention is based, at least in part, on the discovery thatcompositions and methods for primary cells in a high-throughputco-culture platform, image analysis for distinguishing cells inco-cultures and assays that are suitable for screening of agents inepithelial cells, such as hepatocytes.

In one aspect, the invention includes a co-culture for high throughputanalysis of primary hepatocytes comprising a layer of feeder cellsdisposed in a well of a microtiter plate, a layer of primary hepatocytesdisposed on the feeder cells at a concentration that prevents contactinhibition of the hepatocytes, and an amount of culture media thatsupports the hepatocytes and maintains at least one hepatocytebiological activity, wherein the amount is optimized to balance oxygentransport and nutrient supply.

In another aspect, the invention includes a method for high throughputdetection of primary epithelial cells in co-culture, comprisingproviding a co-culture present in a microtiter plate, wherein theco-culture comprises feeder cells and primary epithelial cells,acquiring and comparing images of cell nuclei using a high-throughputscreening microscope, thereby detecting primary epithelial cells inco-culture.

In yet another aspect, the invention includes a method for detectingprimary epithelial cell proliferation or cell death in co-culture,comprising providing a co-culture present in a microtiter plate, whereinthe co-culture comprises feeder cells and primary epithelial cells,acquiring and comparing images of cell nuclei at a first and a secondtime point using a high-throughput screening microscope, and comparingthe number of primary epithelial cell nuclei present at the first andsecond time points, wherein an increase in the number of epithelial cellnuclei present at the second time point detects an increase inepithelial cell proliferation, and detection of a decrease in primaryepithelial cell nuclei present at the second time point detects anincrease in cell death.

In still yet another aspect, the invention includes a method fordetecting an agent that increases primary epithelial cell proliferation,comprising contacting a co-culture present in a microtiter plate with anagent, wherein the co-culture comprises feeder cells and primaryepithelial cells, acquiring and comparing images of primary epithelialcell nuclei at a first and a second time point using a high-throughputscreening microscope, and detecting an increase in the number of primaryepithelial cell nucleic present in the contacted co-culture relative toan untreated co-culture, wherein detection of an increase in the numberof primary epithelial cell nucleic present in the contacted co-cultureidentifies an agent that increases primary epithelial cellproliferation.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the invention includes the microtiter platecomprising at least 384-wells.

In another embodiment, the hepatocyte biological activity is selectedfrom the group consisting of albumin secretion, liver-specific proteinsynthesis, bile production, detoxification of compounds, energymetabolism, and cholesterol metabolism.

In some embodiments, the hepatocytes and feeder cells are plated at aratio of 1:4. In another embodiment, the feeder cells and hepatocytesare of different species. In yet another embodiment, the feeder cellsare present as a confluent layer without aggregation. In still yetanother embodiment, the feeder cells express a protein selected from thegroup consisting of Delta-like homolog 1; C-fos-induced growth factor;Ceruloplasmin; Decorin; Interferon regulatory factor 1; 204interferon-activatable protein; Splicing factor, arginine/serine-rich 3;JKTBP; Autoantigen La; High mobility group box 1; Esk kinase; mousedihydrofolate reductase gene: 3′ end; Pm1 protein; and RacGTPase-activating protein 1.

In another embodiment, the feeder cells comprise one or more types ofnon-parenchymal cells, such as fibroblast or fibroblast-derived cells,and hepatic non-parenchymal cells. The hepatic non-parenchymal cells canbe selected from the group consisting of Kupffer cells, Ito cells,endothelial cells, stellate cells, cholangiocytes, and hepatic naturalkiller cells. In yet another embodiment, the cell adhesion substrate isselected from the group consisting of collagen, fibronectin,vitronectin, laminin, entactin, Arg-Gly-Asp (RGD) peptide,Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide, glycosaminoglycans (GAGs),hyaluronic acid (HA), integrins, ICAMs, selectins, cadherin, andcell-surface protein-specific antibodies, or a combination thereof.

In another embodiment, the culture media comprises hydrocortisone.

In some embodiments, invention includes comparing images of cell nucleicomprises comparing nuclear size, shape, intensity, proximity, andtexture, thereby distinguishing feeder cells from primary epithelialcells.

In one embodiment, each well of the microtiter plate comprises at leastabout 10-500 microliters of liquid or at least about 15-145 microlitersof liquid.

In another embodiment, the primary epithelial cells comprisehepatocytes.

In yet another embodiment, the invention further includes detectingwhether hepatocytes in the microtiter plate retain hepatocyte identityby measuring hepatocyte biological activity. The hepatocyte biologicalactivity is measured using an immunoassay that detects albumin output asa surrogate marker for protein synthesis, using a colorimetric assaythat detects urea generation as a surrogate marker for amino acidmetabolism function, or by detecting cytochrome P450 activity as asurrogate marker for detoxification.

Another aspect of the invention includes a method for optimizing aco-culture of primary hepatocytes for use in the method describedherein, the method comprising plating primary hepatocytes and feedercells into wells of a microtiter plate at about a 1:4 ratio, whereineach well comprises at least about 10-150 microliters of culture media.

Yet another aspect of the invention includes a method for distinguishingtwo or more cell types in a co-culture comprising imaging nuclei of thetwo or more cell types, and comparing nuclear morphology of the nucleito distinguish the cell types.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the invention includes the nuclearmorphology comprising at least one selected from the group consisting ofnuclear size, nuclear shape, nuclear intensity, nuclear proximity, andnuclear texture. In another embodiment, the invention includes producingcomputer images of the nuclei, such as by automatically calculating anumber of nuclei of individual cell types in the co-culture. In anotherembodiment, the invention includes acquiring two or more images atsuccessive time points, such as by quantifying a change in nucleinumbers of individual cell types in the co-culture.

In another embodiment, the invention includes quantifying nucleiundergoing mitosis, such as by quantifying metaphase and anaphasenuclei.

In another aspect, the invention includes a method for assessing anagent that alters hepatocyte biological activity, comprising contactinga hepatocyte present in the co-culture of any one of claims 1-12 with anagent, and assaying for an alteration in hepatocyte biological activityrelative to a control hepatocyte not exposed to the agent, whereindetection of the alteration identifies the agent as altering hepatocytebiological activity.

In yet another aspect, the invention includes a method for assessing themetabolism of a test agent by hepatocytes comprising exposing theco-culture of any one of claims 1-12 to a test agent, and detecting,identifying, and/or quantifying metabolites of the test agent.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the invention includes the hepatocytebiological activity is proliferation, viability, differentiation,toxicity, or cell death. In another embodiment, the invention furthercomprises measuring albumin output as a surrogate marker for proteinsynthesis; measuring urea generation as a surrogate marker for aminoacid metabolism function; and/or measuring cytochrome P450 activity as asurrogate marker for detoxification.

The present invention provides a co-culture system and assays compatiblewith automated high throughput detection and/or quantification ofcellular activity in response to agents and/or environmental conditionsin epithelial cells. The present invention further provides methods forassessing the effects of agents on epithelial cells and predicting theeffect of a test agent on epithelial cells of a subject in vivo. Thepresent invention further provides a method for assessing the metabolismof a test agent by hepatocytes, e.g., by hepatocytes of a particularsubject to be treated with the test agent. In preferred embodiments ofthe various compositions, cultures, and methods disclosed herein, theepithelial cells are hepatocytes, such as human hepatocytes or primaryhuman hepatocytes.

The invention provides a co-culture comprising i) a surface coated by acell adhesion substrate; ii) a layer of feeder cells disposed on thecell adhesion substrate; and iii) a layer of epithelial cells disposedon the opposite surface of the feeder cells relative to the celladhesion substrate. The epithelial cells may comprise human epithelialcells or primary human epithelial cells.

In certain embodiments, the feeder cells comprise non-parenchymal cellsor cells (such as fibroblasts) expressing one or more proteins selectedfrom Table 1. Non-parenchymal cells may comprise stromal cells orhepatic non-parenchymal cells. Stromal cells may comprise fibroblast orfibroblast-derived cells, such as murine, embryonic, or murine embryonicJ2-3T3 fibroblasts. In certain embodiments, hepatic non-parenchymalcells are selected from Kupffer cells, Ito cells, endothelial cells,stellate cells, cholangiocytes (bile duct cells), and hepatic naturalkiller cells (pit cells).

In certain embodiments, feeder cells are growth-inhibited. In certainembodiments, the co-culture further comprises hydrocortisone.

In certain embodiments, the layer of feeder cells is confluent.

In certain embodiments, the one or more epithelial cells contact thefeeder cells.

In certain embodiments, the cell adhesion substrate comprises collagen(such as collagen I), fibronectin, vitronectin, laminin, entactin,Arg-Gly-Asp (RGD) peptide, Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide,glycosaminoglycans (GAGs), hyaluronic acid (HA), integrins, ICAMs,selectins, cadherin, or cell-surface protein-specific antibodies, or acombination thereof.

In certain embodiments, the surface is a surface of a culture well orglass slide.

In certain embodiments, the co-culture is housed in a bioreactor. Incertain embodiments, the bioreactor controls gas exchange across thecell populations. In certain embodiments, the bioreactor controls oxygengradient across the cell populations.

In certain embodiments, the invention provides a multiwell platecomprising a plurality of wells containing a co-culture as describedherein, wherein the plate is compatible for use in high-throughputscreening of agents, as well as methods of preparing cultures in suchplates. In certain embodiments, the multiwell plate contains 96, 384, ormore than 384 wells.

In certain embodiments, the invention provides a method of assessing theeffect of a test agent on an epithelial cell, such as a hepatocyte, inthe co-culture of the invention, e.g., by contacting the epithelial cellwith the test agent and assaying for a pharmacological or toxicologicaleffect in the epithelial cells relative to a control epithelial cell nottreated with the test agent. The pharmacological or toxicological effectmay be proliferation, survival, differentiation, toxicity, orcombinations thereof. In certain embodiments, the assay comprisesquantifying the number of epithelial cells in the co-culture of theinvention by using nuclear morphologies to distinguish an epithelialcell from a feeder cell. In certain embodiments, the assay is selectedfrom measuring albumin synthesis, urea secretion, and cytochrome p450activity, or combinations thereof.

In certain embodiments, the invention provides a method for predictingthe effect of a test agent on epithelial cells, such as hepatocytes, ofa subject in vivo, comprising culturing epithelial cells obtained from asubject in the co-culture of the invention, exposing the epithelial cellto the test agent, and assaying for a pharmacological or toxicologicaleffect of the test agent on the epithelial cells relative to controlepithelial cells not treated with the test agent. The pharmacological ortoxicological effect may be proliferation, survival, differentiation,toxicity, or combinations thereof. In certain embodiments, the assaycomprises quantifying the number of epithelial cells in the co-cultureby using nuclear morphologies to distinguish an epithelial cell from afeeder cell. In certain embodiments, particularly where the cell is ahepatocyte, the assay is selected from measuring albumin synthesis, ureasecretion, and cytochrome p450 activity, or combinations thereof.

In certain embodiments, the invention provides a method for assessingthe metabolism of a test agent by epithelial cells, preferablyhepatocytes, comprising exposing the co-culture of the invention to atest agent, and determining the effect of the epithelial cells on thetest agent. For example, the effect may be measured by detecting,identifying, and/or quantifying metabolites of the test agent, or bydetermining the half-life of the test agent in the presence of theepithelial cells.

In certain embodiments, the invention provides a method for predictingthe metabolism of a test agent by epithelial cells, preferablyhepatocytes, of a subject in vivo, comprising culturing epithelial cellsobtained from a subject in the co-culture of the invention, exposing theepithelial cells to the test agent, and determining the effect of theepithelial cells on the test agent. For example, the effect may bemeasured by detecting, identifying, and/or quantifying metabolites ofthe test agent.

In another aspect, the invention provides a method for producing aco-culture as described above. For example, the method may include:

i) coating a surface with a cell adhesion substrate;

ii) culturing a layer of feeder cells on the cell adhesion substrate;and

iii) overlaying one or more epithelial cells, such as hepatocytes, ontothe feeder cells.

The feeder cells and epithelial cells used in the above method can beany of the types of feeder cells and epithelial cells discussed indetail above with respect to the co-culture. The surface may be thesurface of a culture well or glass slide, or any other suitable surface.Co-cultures can be contained in a multiwell plate having a 96-, 384-,1536- or more than 1536-well format.

In certain embodiments, the method comprises culturing the layer offeeder cells to confluence, e.g., prior to introducing epithelial cells.Hydrocortisone or another compound that serves to limit growth and/orproliferation of the feeder cells may be added to the culture,optionally after having cultured the feeder cells to confluence, butpreferably before introduction of the epithelial cells. The feeder cellsmay be cultured for at least for about 24 hours before epithelial cellsare added.

Overlaying one or more epithelial cells may comprise dispersing a sparsepopulation of epithelial cells, such as hepatocytes, on a confluentfeeder cell layer. The method may further comprise maintaining theepithelial cells for at least 7 days after overlaying the epithelialcells on the feeder cells prior to using the culture for any of themethods discussed herein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “cell” is meant a structural unit of tissue of a multicellularorganism in a living body which is surrounded by a membrane structurewhich isolates it from the outside and has genetic information and amechanism for expressing the genetic information. Cells used herein maybe naturally-occurring cells or artificially modified cells (e.g.,fusion cells, genetically modified cells, etc.).

As used herein, “cellular differentiation” or “differentiation” is theprocess by which a less specialized cell becomes a more specialized celltype.

By “computer image-based readout” is meant numerical cell measurementsderived from a computerized image taken of the co-culture. Platformreadiness can be assessed via statistical parameters such as Z′-factor,which reflects the confidence (Z′>0) in the assay readout by detectingboth assay signal dynamic range and variation, and is mathematicallydefined:

$Z^{\prime} = {1 - \frac{\left( {{3\sigma_{c +}} + {3\sigma_{c -}}} \right)}{{\mu_{c +} - \mu_{c -}}}}$where “c+”=positive control, “c−”=negative control, “σ”=standarddeviation and “μ”=average. Assuming normal distribution, assays withpositive Z′-factors can separate 99.8% of the negative and positivecontrol populations (i.e., the two populations, as defined by meansignal+/−3 standard deviations, do not overlap), essentially separatingsignal from noise.

By “contact inhibition” is meant the cessation of cellular growth,movement, growth processes, or division, upon contact with another cell.

By “culture media” is meant the growth medium with nutrients that isdesigned to support the growth of cells. The culture media can bespecialized for a specific cell type or specific cell process, such asgrowth and proliferation as opposed to differentiation or maturation ofthe cells.

By “amount of culture media” is meant the optimal amount of liquid thatsupports expansion of the cells while allowing gas and nutrientexchange. The amount of culture media is optimized for the cells and theculture format. For example, larger cells in a multi-well formatrequires more media per cell than smaller cells. Additionally, smallmulti-well formats, such as 384 wells or smaller, require less media perwell than larger multi-well formats, such as a 12 wells or 48 wells.

By “epithelial cell” is meant a cell that lines a body cavity or organand/or covers an external surface of an organ. Epithelial cells maintaina closed barrier to the external environment and provide the first lineof defense against disease or infection. Examples of epithelial cellsinclude, hepatocytes, alveolar cells, skin epithelia, gastrointestinaltract lining, mucus lining, and lining of vessels and capillaries.

By the term “feeder cells” is meant cells that are usually adherent andgrowth-arrested but viable and bioactive. Feeder cells provide an intactand functional extracellular matrix and secrete matrix-associatedfactors and cytokines. Feeder cells are typically used to support thegrowth and survival of a second cell type. Examples of feeder cellsinclude, but are not limited to, non-parenchymal cells, such asfibroblast or fibroblast-derived cells. Exemplary examples forhepatocyte cultures may include, hepatic non-parenchymal cells, such asKupffer cells, Ito cells, endothelial cells, stellate cells,cholangiocytes (bile duct cells), and hepatic natural killer cells (pitcells).

The term “hepatocyte” as used herein is meant to include hepatocyte-likecells that exhibit some but not all characteristics of maturehepatocytes, as well as mature and fully functional hepatocytes. Thecells produced by this method may be as at least as functional as thehepatocytes produced by directed differentiation to date. This techniquemay, as it is further improved, enable the production of completelyfully functional hepatocytes, which have all characteristics ofhepatocytes as determined by morphology, marker expression, in vitro andin vivo functional assays.

By “hepatocyte characteristic” is meant a feature or quality that isdisplayed by hepatocyte cells. Typically, the hepatocyte characteristicis specific to the hepatocyte. Examples of hepatocyte characteristicsinclude, but are not limited to, hepatocyte surface markers, distinctnuclei, polygonal morphology, well-demarcated cell-cell borders, andvisible bile canaliculi network.

By “hepatocyte biological activity” is meant an activity or process thatis specific to hepatocytes. Examples of hepatic function include, butare not limited to, liver-specific protein synthesis, albumin secretion,bile production, detoxification of compounds, energy (amino acids, fats,sugars etc.) metabolism, and cholesterol metabolism.

By “high throughput detecting” and “high throughput detection” refers toa process that uses a combination of modern robotics, data processingand control software, liquid handling devices, and/or sensitivedetectors, to efficiently process a large amount of (e.g., thousands,hundreds of thousands, or millions of) samples in biochemical, geneticor pharmacological experiments, either in parallel or in sequence,within a reasonably short period of time (e.g., days). Preferably, theprocess is amenable to automation, such as robotic simultaneous handlingof 96 samples, 384 samples, 864 samples, 1536 samples or more. A typicalhigh throughput screening robot tests up to 100,000 to a few hundredthousand compounds per day. The samples are often in small volumes, suchas no more than 1 mL, 500 μl, 200 μl, 100 μl, 50 μl or less.

“High-throughput screening,” “high throughput screen” and “HTS” refersto a process that uses high throughput detection. A typical HTS robottests up to 100,000 to a few hundred thousand compounds per day. Thesamples are often in small volumes, such as no more than 1 mL, 500 μl,200 μl, 100 μl, 50 μl or less. Through this process one can rapidlyidentify active compounds, small molecules, antibodies, proteins orpolynucleotides which modulate a particular biomolecular/geneticpathway. The results of these experiments provide starting points forfurther drug design and for understanding the interaction or role of aparticular biochemical process in biology. Thus “high-throughputscreening” as used herein does not include handling large quantities ofradioactive materials, slow and complicated operator-dependent screeningsteps, and/or prohibitively expensive reagent costs, etc.

By “high throughput screening microscope” is meant a microscopeconfigured to be self-focusing. Optionally, the high throughputscreening microscope is coupled to a barcode reader and robotic arm forautomated plate loading. The high-throughput screening microscope hasthe capacity to view fluorescently labeled samples.

By “liver condition” is meant a disease, disorder, or condition with oraffecting the liver. The liver condition affects or disrupts liverfunction, such as storing and filtering blood, liver-specific proteinsynthesis, bile production, detoxification of compounds, energymetabolism, and cholesterol metabolism.

By “miniaturized high through-put assay” is meant a small-scale cultureformat. In an exemplary embodiment, the miniaturized high through-putassay is a multi-well format having at least 384 wells, 864 well, 1534wells, etc. By “microtiter plate” is meant a small scale culture formatcomprising. Examples of such multi-well formats include, but are notlimited to, 96-well, 384-well, 864-well, 1536-well or greater than1536-well format.

By “multi-well format” is meant a culture format comprising more thanone well. Examples of such multi-well formats include, but are notlimited to, 6-well, 12-well, 24-well, 48-well, 96-well, 384-well,864-well, 1536-well or greater than 1536-well format.

By “non-aggregated” or “without aggregation” is meant distributing thecells as discrete cells with sufficient room to proliferate, not ascolonies of cells in a small area.

By “nuclear morphology” is meant the features of nuclei associated witha specific cell type, species, stage of development, diseased status,etc. Nuclear morphology can be assessed by the shape, size, granularity,intensity, proximity, and/or texture of nuclei. Differences in nuclearmorphologies can be useful in distinguishing cell types, species, stagesof development, or disease status.

By “surrogate marker” is meant a measurement of an activity or biomarkerthat indicates, reflects or substitutes for another measurement of anactivity or biomarker.

By “agent” is meant any small molecule chemical compound, antibody,nucleic acid molecule, or polypeptide, or fragments thereof.

By “ameliorate” is meant decrease, suppress, attenuate, diminish,arrest, or stabilize the development or progression of a disease.

By “cell adhesion substrate” is meant a molecule or compound that aidsin cell adhesion. Examples of cell adhesion substrates include, but arenot limited to, extracellular matrix proteins, collagen, fibronectin,vitronectin, laminin, entactin, Arg-Gly-Asp (RGD) peptide,Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide, glycosaminoglycans (GAGs),hyaluronic acid (HA), integrins, ICAMs, selectins, cadherin, andcell-surface protein-specific antibodies, or a combination thereof.

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. Patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. Patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

“Detect” and “detecting” refers to identifying or measuring thepresence, absence or amount of a biological activity or cell.

By “detectable label” is meant a composition that when linked to amolecule of interest renders the latter detectable, via spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radioactive isotopes, magnetic beads,metallic beads, colloidal particles, fluorescent dyes, electron-densereagents, enzymes (for example, as commonly used in an ELISA), biotin,digoxigenin, or haptens.

By “disease” is meant any condition or disorder that damages orinterferes with the normal function of a cell, tissue, or organ.Examples of diseases include, but are not limited to, liver-basedmetabolic disease, chronic liver failure, acute liver failure, geneticmetabolic defect, familial tyrosinemia, cirrhosis, hepatitis, liverabscesses and drug induced liver failure.

By “effective amount” is meant the amount of a required to amelioratethe symptoms of a disease relative to an untreated patient. Theeffective amount of active compound(s) or cells used to practice thepresent invention for therapeutic treatment of a disease variesdepending upon the manner of administration, the age, body weight, andgeneral health of the subject. Ultimately, the attending physician orveterinarian will decide the appropriate amount and dosage regimen. Suchamount is referred to as an “effective” amount.

The invention provides a screening assay and detection method that areuseful for the discovery of drugs to induce expansion, differentiationor antagonize processes that induce expansion and/or differentiation ofcells, such as liver cells. In addition, the methods of the inventionprovide a facile means to identify therapies that are safe for use insubjects. In addition, the methods of the invention provide a route foranalyzing virtually any number of compounds for effects on a celldescribed herein with high-volume throughput, high sensitivity, and lowcomplexity.

“Embryonic stem (ES) cells” are pluripotent stem cells derived fromearly embryos. An ES cell was first established in 1981, which has alsobeen applied to production of knockout mice since 1989. In 1998, a humanES cell was established, which is currently becoming available forregenerative medicine.

Unlike ES cells, tissue stem cells have a limited differentiationpotential. Tissue stem cells are present at particular locations intissues and have an undifferentiated intracellular structure. Therefore,the pluripotency of tissue stem cells is typically low. Tissue stemcells have a higher nucleus/cytoplasm ratio and have few intracellularorganelles. Most tissue stem cells have low pluripotency, a long cellcycle, and proliferative ability beyond the life of the individual.Tissue stem cells are separated into categories, based on the sites fromwhich the cells are derived, such as the dermal system, the digestivesystem, the bone marrow system, the nervous system, and the like. Tissuestem cells in the dermal system include epidermal stem cells, hairfollicle stem cells, and the like. Tissue stem cells in the digestivesystem include pancreatic (common) stem cells, liver stem cells, and thelike. Tissue stem cells in the bone marrow system include hematopoieticstem cells, mesenchymal stem cells, and the like. Tissue stem cells inthe nervous system include neural stem cells, retinal stem cells, andthe like.

“Induced pluripotent stem cells,” commonly abbreviated as iPS cells oriPSCs, refer to a type of pluripotent stem cell artificially preparedfrom a non-pluripotent cell, typically an adult somatic cell, orterminally differentiated cell, such as fibroblast, a hematopoieticcell, a myocyte, a neuron, an epidermal cell, or the like, by insertingcertain genes, referred to as reprogramming factors.

As used herein, the term “stem cell” refers to a cell capable of givingrising to at least one type of a more specialized cell. A stem cell hasthe ability to self-renew, i.e., to go through numerous cycles of celldivision while maintaining the undifferentiated state, and has potency,i.e., the capacity to differentiate into specialized cell types.Typically, stem cells can regenerate an injured tissue. Stem cellsherein may be, but are not limited to, embryonic stem (ES) cells,induced pluripotent stem cells, or tissue stem cells (also calledtissue-specific stem cell, or somatic stem cell). Any artificiallyproduced cell which can have the above-described abilities (e.g., fusioncells, reprogrammed cells, or the like used herein) may be a stem cell.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, the cellsof this invention are purified if they are substantially free of othercells, viral material, or other components. Purity and homogeneity aretypically determined using analytical techniques, for example, flowcytometry.

By “surface marker” is meant any protein or carbohydrate found on thesurface of a cell that can be detected by immunological staining, flowcytometry, ELISA, or other assays known to those having ordinary skillin the art. The surface marker may also be associated with expressionlevel or activity or alteration in expression or activity that isassociated with particular cell type, stage of development, a disease ordisorder.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, isolating, purifying or otherwise acquiringthe agent.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%,75%, or 100%.

By “reference” is meant a standard or control condition.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or symptoms associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder, condition orsymptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of a 384-well co-culture platform.

FIG. 1B is a graph showing fibroblast-mediated hepatocyte stabilizationfor at least 9 days. Line graph shows representative rate of albuminsecretion in screening co-cultures and hepatocyte-only cultures (green)over time. All data presented mean±standard deviation.

FIG. 2 shows cryopreserved primary human hepatocytes were maintained invitro through co-cultivation upon a feeder layer of J2-3T3 fibroblastsin 384-well formats. Phase contrast imaging shows morphology offeeder-layer co-cultures (scale bar=100 um).

FIG. 3 is a panel of images showing distinctive nuclei morphology.Hepatocytes in co-culture with J2-3T3 fibroblasts were distinguishedbased on nuclei morphology. Hepatocyte nuclei (left) were smaller,rounder and more uniform in texture while fibroblast nuclei (right) werelarger, more elliptical, and more punctate. Differences in species andcell type of the hepatocyte and fibroblast may account for differencesin nuclei morphology.

FIGS. 4A-4D shows the image-based assay workflow. FIG. 4A shows thatnuclei were visualized with Hoechst stain, and imaged using ahigh-content screening microscope.

FIG. 4B is an image detailing the differences in nuclear morphologies ofHoechst stained hepatocyte and fibroblast nuclei.

FIG. 4C is an image showing the user interface window to identify andcharacterize nuclei through a custom image-based proliferation assay.The user interface window of the classification software, CellProfilerAnalyst, is shown. It allowed manual classification of randomlypresented nuclei and error correction of machine-classified nuclei.

FIG. 4D shows an example of the automated nuclei classification andcounting analysis generated by the software.

FIG. 5 is a panel of images showing uniformity of image intensitythroughout the screen. Permeabilization treatment was not necessary fortraditional Hoechst staining but helped normalize Hoechst 33258 stainingintensities throughout screening. Upper panel shows heatmap of imageintensities for each 384-well plate; arrows indicate location ofbrightest and dimmest images. Bottom panel shows acquired images.

FIG. 6 is a schematic representation of the automated image acquisition.Treated sample plates were robotically loaded into a high-throughputscreening microscope.

FIG. 7 shows representative images of nuclei identification. The feederlayer co-culture led to overlapping objects in Hoechst images thatproved challenging to segment. The final algorithm was able to correctlyidentify nuclei locations and borders.

FIG. 8 shows images of sub-nuclear structure identification. Punctatesub-nuclear structures were identified as objects and associated withtheir parent nucleus. Circles indicate hepatocyte islands. The squaresurrounds one region of fibroblast cluster.

FIG. 9 is a schematic representation showing the classificationaccuracy. Screening images were classified without (left) and with(right) the identification of punctate sub-nuclear structures. Thesquares indicate fibroblast nuclei that were erroneously identified ashepatocyte nuclei.

FIG. 10 is an image showing mitotic nuclei morphology. The left squaremarks a nucleus with morphology consistent with metaphase; the rightsquare marks a nucleus with morphology consistent with anaphase.

FIG. 11A is a graph showing biochemical functional assay for albuminsecretion as a function of hepatocyte density in screening cultures. Alldata are presented as mean±standard deviation.

FIG. 11B is a graph showing biochemical functional assay for ureaproduction as a function of hepatocyte density in screening cultures.All data are presented as mean±standard deviation.

FIG. 11C is a graph showing biochemical functional assay for cytochromeP450 activity as a function of hepatocyte density in screening cultures.All data are presented as mean±standard deviation.

FIG. 12 is a schematic representation of a competitive ELISA.

FIG. 13 is a series of graphs and high content images which show thatthe screening platform stabilized hepatocyte phenotypic function invitro. The bar graph shows albumin secretion as a function of hepatocytedensity in screening cultures. The inset above the bar graph is aphase-contrast image showing the morphology of the feeder-layercocultures (scale bar, 100 μm). Hoechst staining of screening coculturesshowing that hepatocyte nuclei (four left circles) have a uniformtexture, whereas fibroblast nuclei (four right circles) are punctate(scale bars, 50 μm). An automated high-content imaging assay identifiesand classifies individual nuclei.

FIG. 14 is a summary of the workflow of the primary screening of smallmolecules.

FIG. 15 is a series of graphs showing the type of compounds thatconstituted the initial set of 93 compounds that met all hit selectioncriteria qualifying as proliferation hits (FPH) and also scatterplots ofprimary screening data (Example 1). The bar graph shows categories ofscreened and hit compounds. The scatter plots display replicates of thescreen, shown separately for the image-based proliferation andcompetitive ELISA functional readouts. Data points represent DMSO andexperimental small molecules. The boxed regions indicate hit zones. Chr,chromatin-biased compounds; Com, commercially available compounds; Dos,products of diversity-oriented synthesis; Kin, kinase-biased compounds;Bio, compounds with previously documented bioactivity; Nat, naturalproducts; HepCount, count of hepatocyte nuclei.

DETAILED DESCRIPTION OF THE INVENTION

As described below, the present invention features assays forco-culturing primary cells while maintaining key biological activitiesspecific to the primary cells. The invention is based, at least in part,on the discovery that compositions and methods for primary cells in ahigh-throughput co-culture platform, image analysis for distinguishingcells in co-cultures and assays that are suitable for screening ofagents in epithelial cells, such as hepatocytes.

Co-Culture

The present invention includes a co-culture for high throughput analysisof primary hepatocytes comprising a layer of feeder cells disposed in awell of a microtiter plate, a layer of primary hepatocytes disposed onthe feeder cells at a concentration that prevents contact inhibition ofthe hepatocytes, and an amount of culture media that supports thehepatocytes and maintains at least one hepatocyte biological activity,wherein the amount is optimized to balance oxygen transport and nutrientsupply

The invention includes a co-culture optimized for a multi-well format.The multi-well format includes larger formats, such as 6, 12, 24, 48,and 96 wells. In one embodiment, the feeder cells and hepatocytes aredisposed in a microtiter plate. The microtiter plate includes thosehaving at least 384, 864, 1536 wells, or a greater number of wells.

In another embodiment, the concentration of hepatocytes disposed on thefeeder cells is at an optimal concentration that preventscontact-inhibition of the hepatocytes. The hepatocytes can be plated ata ratio to the feeder cells of less than about 1:4. In some embodiments,the concentration comprises a ratio of hepatocytes to the feeder cellsof less than about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or anyratio therebetween.

The co-culture further maintains hepatocyte functions andcharacteristics of the hepatocytes. In one embodiment, the hepatocytesmaintain at least one hepatocyte biological activity throughoutco-culturing. Examples of hepatocyte biological activity include, butare not limited to, liver-specific protein synthesis (albuminsecretion), bile production, detoxification of compounds, energy (aminoacid, fat and sugar) metabolism, and cholesterol metabolism.

In certain embodiments, the co-culture comprises i) a surface coated bya cell adhesion substrate; ii) a layer of feeder cells disposed on thecell adhesion substrate; and iii) a layer of epithelial cells, such ashepatocytes, disposed on the opposite surface of the feeder cellsrelative to the cell adhesion substrate. The epithelial cells maycomprise human epithelial cells, primary human epithelial cells,endothelial-derived epithelial cells, or hepatocytes, includinghepatocytes expanded in animals (e.g., as produced in mice, such ascells available from Yecuris Corporation as human hepatocytes) andhepatocytes derived from adult stem cells, embryonic stem cells, orinduced pluripotent stem cells such as iHep cells.

In certain embodiments, iHep cells are derived from induced pluripotentstem cells by i) culturing undifferentiated iPS cells on a hydrogelprotein matrix; ii) transferring confluent iPS cells to differentiationmedia; and iii) adding growth factors (Activin A, BMP-4, bFGF, HGF, andOSM).

In certain embodiments, epithelial cells are obtained from a subjectwith a healthy organ, such as hepatocytes from a healthy liver, while inother embodiments the epithelial cells may be obtained from a subjectwith a diseased organ, such as hepatocytes, from a disease liver. Theepithelial cells may be adult or embryonic.

Feeder cells are important to maintain the quality of the epithelialcells (such as hepatocytes) in the culture. Suitable feeder cells maycomprise non-parenchymal cells or cells (such as fibroblasts) expressinga protein selected from Table 1 or Delta-like homolog 1; C-fos-inducedgrowth factor; Ceruloplasmin; Decorin; Interferon regulatory factor 1;204 interferon-activatable protein; Splicing factor,arginine/serine-rich 3; JKTBP; Autoantigen La; High mobility group box1; Esk kinase; mouse dihydrofolate reductase gene: 3′ end; Pm1 protein;and Rac GTPase-activating protein 1.

TABLE 1 Fibroblast Candidate Genes Whose Expression Profiles CorrelatePositively With the Inductive Profile Shown in FIG. 1A Accession NumberDescription Cell Surface Z12171 Delta-like homolog 1 (Drosophila)Secreted X99572 C-fos-induced growth factor (VEGF-D) U49513 Smallinducible cytokine A9 U49430 Ceruloplasmin Extracellular matrix ormatrix remodeling X53929 Decorin Transcription factors M21065 Interferonregulatory factor 1 M31419 204 interferon-activatable protein OtherX53824 Splicing factor, arginine/serine-rich 3 AB017020 Heterogeneousnuclear ribonucleoprotein D-like protein JKTBP L00993 Autoantigen La(SS-B) U004311 High mobility group box Z72486 DNA polymerase delta smallsubunit (pold2) M86377 Esk kinase J00388 Mouse dihydrofolate reductasegene: 3′ end X07967 Pm1 protein AW122347 (EST) Rac GTPase-activatingprotein 1 AA655369 (EST) Translocase of inner mitochondrial membrane 8homolog a, yeast NOTE. Unknown function EST accession numbers; AI037577,AI846197, AI841894, AI606951, AA940036, AI746846, AI551087, AA222883,AI848479.

In certain embodiments, wherein feeder cells comprise non-parenchymalcells, non-parenchymal cells may comprise stromal cells or hepaticnon-parenchymal cells. In certain embodiments, wherein non-parenchymalcells comprise stromal cells, stromal cells may comprise fibroblast orfibroblast-derived cells. In certain embodiments, wherein stromal cellscomprise fibroblast or fibroblast-derived cells, fibroblast orfibroblast-derived cells may be murine and/or embryonic. In a preferredembodiment, the feeder cells are murine embryonic J2-3T3 fibroblasts.

In certain embodiments, wherein non-parenchymal cells comprise hepaticnon-parenchymal cells, hepatic non-parenchymal cells are selected fromKupffer cells, Ito cells, endothelial cells, stellate cells,cholangiocytes (bile duct cells), and hepatic natural killer cells (pitcells).

In some embodiment, the feeder cells have different morphologies and/orcharacteristics from the hepatocytes. For example, the feeder cells mayhave different nuclear morphology, be of a different species (e.g.,mouse vs. human), and a different cell type (non-parenchymal vsepithelial cells).

In certain embodiments, feeder cells are growth-inhibited, which helpsavoid overgrowth of the feeder cells and maintain confluent feeder cellsin a single layer. In one embodiment, the feeder cells are present as aconfluent layer without aggregation. Feeder cells can begrowth-inhibited by irradiation, treatment with mitomycin c,high-temperature treatment, chemical fixation, treatment with steroidssuch as hydrocortisone or dexamethasone, or any other suitable meansthat reduces their proliferative capacity.

In certain embodiments, the co-culture further comprises hydrocortisone,which also helps to avoid over-proliferation of the feeder cells. Feedercells growth-inhibited by hydrocortisone appear to support epithelialcells, such as hepatocytes, in the co-culture for an extended period oftime, for example for at least 9 days in culture.

In certain embodiments, the layer of feeder cells is confluent.

In certain embodiments, cell adhesion substrate may be selected fromcollagen type I, collagen type II, collagen type IV, fibronectin,vitronectin, laminin, entactin, Arg-Gly-Asp (RGD) peptide,Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide, glycosaminoglycans (GAGs),hyaluronic acid (HA), integrins, ICAMs, selectins, cadherin, andcell-surface protein-specific antibodies, or a combination thereof. Celladhesion substrates may also be selected from collagen III, collagen IV,collagen V, laminin á2, tenascin-R, chondroitin sulfate proteooglycans,aggrecan, elastin, keratin, mucin, superfibronectin, F-spondin,nidogen-2, heparan sulfate proteoglycan (perlecan), biglycan, decorin,galectin-1, galectin-3, galectin-3c, galectin-4, galectin-8,thrombospondin-4, osteopontin, osteonectin, testican 1, testican 2,fibrin, tenascin-C, nidogen-1, agrin, hyaluronan, and brevican asdisclosed in Reticker-Flynn Nature Communications July 2012; DOI:10.1038/ncomms2128, which is hereby incorporated by reference in itsentirety. Cell adhesion substrates may also be selected from nucleicacids, nucleic acid binding partners, receptors, antibodies, enzymes,carbohydrates, oligosaccharides, polysaccharides, cells, cellaggregates, cell components, lipids, arrays of ligands (e.g.,non-protein ligands), liposomes, microorganisms, e.g., bacteria,viruses, and the like, as disclosed in greater detail in WO2002/04113,which is hereby incorporated by reference in its entirety. In apreferred embodiment, the cell adhesion substrate is collagen I. Coatingthe surface with a cell adhesion substrate fosters secure cellattachment, important for maintenance of co-culture and screeningpurposes.

In certain embodiments, the surface of the co-culture consists ofpolymeric materials, glass, semiconductors, or metals that may bearranged in a variety of configurations, for example a polymeric culturewell or glass slide, or any other suitable combinations thereof. As iswell known in the art, culture wells may be in a single, multi-wellformat or microtiter plate, Multi-well format includes 6-well, 12-well,24-well, 48-well, 96-well, 384-well, 864-well, 1536-well or greater than1536-well format. Microtiter plates include 96-well, 384-well, 864-well,1536-well or greater than 1536-well format.

In certain embodiments, one or more epithelial cells contact the feedercells.

In certain embodiments, the co-culture is housed in a bioreactor, suchas a bioreactor disclosed in WO 2004/076647, which is herebyincorporated by reference in its entirety. In certain embodimentswherein the co-culture is housed in a bioreactor, the bioreactorcontrols gas exchange across the cell populations. In certainembodiments wherein the bioreactor controls gas exchange across the cellpopulations, the bioreactor controls oxygen gradient across the cellpopulations.

In certain embodiments, the co-cultures contains an epithelial cell,such as a hepatocyte, enabling single cell analysis. In otherembodiments, the co-culture contains more than one epithelial cell,enabling multicell analysis.

In certain embodiments, the invention provides a method for producingthe co-culture of the present invention, the method comprising i)coating a surface with a cell adhesion substrate; ii) culturing a layerof feeder cells on the cell adhesion substrate; and iii) overlaying oneor more epithelial cells, such as hepatocytes, onto the feeder cells.

In certain embodiments, the surface of the co-culture consists ofpolymeric materials, glass, semiconductors, or metals that may bearranged in a variety of configurations, for example a polymeric culturewell or glass slide, or any other suitable combinations thereof. As iswell known in the art, culture wells may be in a single or multiwellformat, such as a 96-well, 384-well, 1536-well or greater than 1536-wellplate.

In certain embodiments, cell adhesion substrate may be selected fromcollagen type I, collagen type II, collagen type IV, fibronectin,vitronectin, laminin, entactin, Arg-Gly-Asp (RGD) peptide,Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide, glycosaminoglycans (GAGs),hyaluronic acid (HA), integrins, ICAMs, selectins, cadherin, andcell-surface protein-specific antibodies, or a combination thereof. Celladhesion substrates may also be selected from collagen III, collagen IV,collagen V, laminin á2, tenascin-R, chondroitin sulfate proteooglycans,aggrecan, elastin, keratin, mucin, superfibronectin, F-spondin,nidogen-2, heparan sulfate proteoglycan (perlecan), biglycan, decorin,galectin-1, galectin-3, galectin-3c, galectin-4, galectin-8,thrombospondin-4, osteopontin, osteonectin, testican 1, testican 2,fibrin, tenascin-C, nidogen-1, agrin, hyaluronan, and brevican asdisclosed in Reticker-Flynn Nature Communications July 2012 DOI:10.1038/ncomms2128, which is hereby incorporated by reference in itsentirety. Cell adhesion substrates may also be selected from nucleicacids, nucleic acid binding partners, receptors, antibodies, enzymes,carbohydrates, oligosaccharides, polysaccharides, cells, cellaggregates, cell components, lipids, arrays of ligands (e.g.,non-protein ligands), liposomes, microorganisms, e.g., bacteria,viruses, and the like, as disclosed in greater detail in WO2002/04113,which is hereby incorporated by reference in its entirety. In preferredembodiments, the cell adhesion substrate is collagen I, optionallypresented as a coating of collagen, which may be adsorbed onto orotherwise disposed on the surface. Coating the surface with a celladhesion substrate, e.g., by adsorbing collagen onto the surface, suchas by incubating the surface in a solution of collagen, allows forsecure cell attachment, important for maintenance of co-culture andscreening purposes.

Methods of Co-Culturing

The present invention also includes, in one aspect, a method for highthroughput detection of primary epithelial cells in co-culture,comprising providing a co-culture present in a microtiter plate, whereinthe co-culture comprises feeder cells and primary epithelial cells,acquiring and comparing images of cell nuclei using a high-throughputscreening microscope, thereby detecting primary epithelial cells inco-culture.

In another aspect, the invention includes a method for detecting primaryepithelial cell proliferation or cell death in co-culture, comprisingproviding a co-culture present in a microtiter plate, wherein theco-culture comprises feeder cells and primary epithelial cells,acquiring and comparing images of cell nuclei at a first and a secondtime point using a high-throughput screening microscope, and comparingthe number of primary epithelial cell nuclei present at the first andsecond time points, wherein an increase in the number of epithelial cellnuclei present at the second time point detects an increase inepithelial cell proliferation, and detection of a decrease in primaryepithelial cell nuclei present at the second time point detects anincrease in cell death.

In yet another aspect, the invention includes a method for detecting anagent that increases primary epithelial cell proliferation, comprisingcontacting a co-culture present in a microtiter plate with an agent,wherein the co-culture comprises feeder cells and primary epithelialcells, acquiring and comparing images of primary epithelial cell nucleiat a first and a second time point using a high-throughput screeningmicroscope; and detecting an increase in the number of primaryepithelial cell nucleic present in the contacted co-culture relative toan untreated co-culture, wherein detection of an increase in the numberof primary epithelial cell nucleic present in the contacted co-cultureidentifies an agent that increases primary epithelial cellproliferation.

The method for detecting is further optimized with each well of themicrotiter plate comprising at least about 10-500 microliters of liquid.In some embodiment, each well of the microtiter plate comprises at leastabout 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500 microliters, or moreof liquid. In one embodiment, each well of the microtiter platecomprises at least about 15-145 microliters of liquid.

To distinguish feeder cells from primary epithelial cells, nuclear size,shape, intensity, proximity, and texture of the cell nuclei arecompared. Different cell types also can be distinguished from oneanother, such as the primary epithelial cells can be distinguished fromthe feeder cells. In another embodiment, the feeder cells and primaryepithelial cells are from different species.

The method can further comprise detecting whether hepatocytes in themicrotiter plate retain hepatocyte identity by measuring hepatocytebiological activity. The hepatocyte biological activity can be measuredusing an immunoassay that detects albumin output as a surrogate markerfor protein synthesis, using a colorimetric assay that detects ureageneration as a surrogate marker for amino acid metabolism functionand/or detecting cytochrome P450 activity as a surrogate marker fordetoxification.

Another aspect of the invention includes a method for optimizing aco-culture of primary hepatocytes for use in any method described hereincomprising plating primary hepatocytes and feeder cells into wells of amicrotiter plate at about a 1:4 ratio, wherein each well comprises atleast about 10-150 microliters of culture media.

The method for detecting primary epithelial cells in the co-cultureprovides for plating feeder cells onto the cell adhesion substratecoated surface. Feeder cells are important to maintain the quality ofthe epithelial cells in the culture. Suitable feeder cells may comprisenon-parenchymal cells or cells (such as fibroblasts) expressing aprotein selected Delta-like homolog 1; C-fos-induced growth factor;Ceruloplasmin; Decorin; Interferon regulatory factor 1; 204interferon-activatable protein; Splicing factor, arginine/serine-rich 3;JKTBP; Autoantigen La; High mobility group box 1; Esk kinase; mousedihydrofolate reductase gene: 3′ end; Pm1 protein; and RacGTPase-activating protein 1 or from Table 1.

In certain embodiments, wherein feeder cells comprise non-parenchymalcells, non-parenchymal cells may comprise stromal cells or hepaticnon-parenchymal cells. In certain embodiments, wherein non-parenchymalcells comprise stromal cells, stromal cells may comprise fibroblast orfibroblast-derived cells. In certain embodiments, wherein stromal cellscomprise fibroblast or fibroblast-derived cells, fibroblast orfibroblast-derived cells may be murine and/or embryonic. In a preferredembodiment, the feeder cells are murine embryonic J2-3T3 fibroblasts. Incertain embodiments, wherein non-parenchymal cells comprise hepaticnon-parenchymal cells, hepatic non-parenchymal cells are selected fromKupffer cells, Ito cells, endothelial cells, stellate cells,cholangiocytes (bile duct cells), and hepatic natural killer cells (pitcells).

In certain embodiments, feeder cells are growth-inhibited, which helpsavoid overgrowth of the feeder cells and maintain confluent feeder cellsin a single layer. Feeder cells are plated onto the cell adhesionsubstrate coated surface and allowed to reach confluence, when theirgrowth becomes contact inhibited. For example, J2-3T3 fibroblasts platedat 8,000 cells/well in a 384-well plate reach confluence 48 hours laterunder typical culture conditions.

In certain embodiments, the co-culture further comprises hydrocortisoneadded to the co-culture medium, which also helps to avoidover-proliferation of the feeder cells. Feeder cells growth-inhibited byhydrocortisone appear to support epithelial cells, such as hepatocytes,in the co-culture for an extended period of time, for example for atleast 9 days.

The method for detecting primary epithelial cells in the co-cultureprovides for plating one or more epithelial cells, such as hepatocytes,onto feeder cells, e.g., such that the epithelial cells contact thefeeder cells. In certain embodiments, a sparse population of epithelialcells is co-cultivated on a confluent feeder cell layer. For example,primary human hepatocytes can be plated onto a confluent layer of J2-3T3fibroblasts on Day 0 at a density below 5,000 cells/well or even below3,000 cells/well, e.g., about 2,000 cells/well, in a 384-well plate, orat a correspondingly low density based on the surface area of the wellin plates of other sizes. The co-culture can then be maintained understandard conditions with daily replacement of medium. This designprovides surface area for cell expansion and stabilizes phenotypicfunctions in vitro.

The epithelial cells may comprise human epithelial cells, primary humanepithelial cells, endothelial-derived epithelial cells, or hepatocytes,including hepatocytes expanded in animals (e.g., as produced in mice,such as human hepatocytes available from Yecuris Corporation) andhepatocytes derived from adult stem cells, embryonic stem cells, orinduced pluripotent stem cells such as iHep cells. In certainembodiments, iHep cells are derived from induced pluripotent stem cellsby i) culturing undifferentiated iPS cells on a hydrogel protein matrix;ii) transferring confluent iPS cells to differentiation media; and iii)adding growth factors (Activin A, BMP-4, bFGF, HGF, and OSM). In certainembodiments, epithelial cells are obtained from a subject with a healthyorgan, such as hepatocytes from a healthy liver, while in otherembodiments the epithelial cells may be obtained from a subject with adiseased organ, such as hepatocytes from a disease liver. The epithelialcells may be adult or embryonic. In preferred embodiments of theforegoing, the epithelial cells are hepatocytes, e.g., obtained from aliver.

Cell Imaging

The present invention includes, in one aspect, a method fordistinguishing two or more cell types in a co-culture comprising imagingnuclei of the two or more cell types, and comparing nuclear morphologyof the nuclei to distinguish the cell types. A key feature of the assayis the ability to distinguish the individual cell types present in theco-culture based on nuclear morphology. The nuclear morphology caninclude nuclear size, shape, intensity, proximity, and texture of thenuclei.

In one embodiment, the invention includes producing computer images ofthe nuclei. The produced computer images can be analyzed to calculatethe number of nuclei of individual cell types. This may be achieved byproducing computer images and automatically calculating a number ofnuclei of individual cell types in the co-culture.

Multiple images of the nuclei can also be acquired. Imaging nuclei caninclude acquiring two or more images at successive time points. Thesuccessive time points can be seconds, minutes, hours, days or weeksapart from one another.

In another embodiment, the invention includes comparing nuclearmorphology by quantifying a change in nuclei numbers of individual celltypes in the co-culture. Comparing nuclear morphology can also includequantifying nuclei undergoing mitosis, including metaphase and anaphasenuclei.

The imaging assay is readily adapted for microscale architectures, suchas microtiter plates having at least 384-wells or more. The co-cultureis also compatible with automated high-throughput screening platforms todetect and/or quantify cellular activity in response to agents and/orenvironmental conditions. For example, the initial density of epithelialcells can be low enough to enable proliferative responses to be assessedin the co-culture. In one embodiment, the epithelial cells are disposedon the feeder cells at a ratio of less than about 1:3.

Although these advantages can be very helpful in a variety ofsituations, culture survival is typically shorter than seen usingco-planar systems where the feeder cells surround pockets ofhepatocytes. However, such co-planar culture systems may limitproliferative expansion due to contact inhibition and therefore are lesswell suited to proliferation studies. Platform readiness for HTS wasassessed via statistical parameters such as z′-factor, which reflectsboth assay signal dynamic range and variation, and is mathematicallydefined:

$Z^{\prime} = {1 - \frac{\left( {{3\sigma_{c +}} + {3\sigma_{c -}}} \right)}{{\mu_{c +} - \mu_{c -}}}}$where “c+”=positive control, “c−”=negative control, “σ”=standarddeviation and “μ”=average. Assuming normal distribution, assays withpositive Z′-factors can separate 99.8% of the negative and positivecontrol populations (i.e., the two populations, as defined by meansignal+/−3 standard deviations, do not overlap), essentially separatingsignal from noise.

In certain embodiments, the assay measures epithelial proliferation byquantifying epithelial nuclei, using nuclear morphologies to distinguishepithelial cells from co-existing feeder cells of a second cell type.For example, hepatic cell nuclei were uniform in texture whilefibroblast nuclei were punctate. Nuclear stains can also be used toenhance visualization and imaging of morphological characteristics. Incertain embodiments, image acquisition is further facilitated by typicalflattening-out phenomena experienced by cells maintained in culture. Incertain embodiments, cells in the feeder layer and in the epitheliallayer of the co-culture exist in the same focal plane, allowingsimultaneous imaging of both layers without the need for refocusing thedetector.

In certain embodiments, a customized, automated, high-content imagingprotocol is used to acquire and analyze images. In certain embodiments,automated image analyses utilize machine learning algorithms to classifynuclei types and tabulate epithelial nuclei numbers. Assay validationdata show that this image-based readout can confidently (z′>0) detectdoublings in hepatic nuclei numbers with low variance (CV<20%) and goodreproducibility for accurate proliferation measurements. In certainembodiments, the number of nuclei in the process of mitosis (i.e.,undergoing metaphase and anaphase) can also be identified andquantified. In certain embodiments, selective labeling of one populationwith lipophilic dyes (i.e., carboxyfluorescein diacetate), nuclearstains (i.e., DAPI and Hoechst), or tagged proteins (i.e., GFP-taggedprotein) can be used to distinguish cells in a population of interestfrom un-labeled cells.

Screening Assays

The present invention also includes assays suitable for high-throughputscreening of agents in epithelial cells, such as hepatocytes. Theco-culture can be used to test the effects of agents on epithelialcells, including predicting the effect of a test agent on epithelialcells of a particular subject. The present invention further provides amethod for assessing the metabolism of a test agent by epithelial cells,including epithelial cells of a particular subject.

The co-culture can be used to screen for agents (such as solvents, smallmolecule drugs, peptides, and polynucleotides) or environmentalconditions (such as culture conditions or manipulation) that affect thecharacteristics of cells. Two or more drugs can be tested in combination(by exposing to the cells either simultaneously or sequentially), todetect possible drug-drug interactions and/or rescue effects (e.g., bytesting a toxin and a potential anti-toxin). Drug(s) and environmentalcondition(s) can be tested in combination (by treating the cells with adrug either simultaneously or sequentially relative to an environmentalcondition), to detect possible drug-environment interaction effects. Useof the co-culture for screening purposes further comprises assays ofcellular activity that include imaging and biochemical read-outs.

In certain embodiments, the assay is selected in a manner appropriate tothe cell type and agent and/or environmental factor being studied asdisclose in WO 2002/04113, which is hereby incorporated by reference inits entirely. For example, changes in cell morphology may be assayed bystandard light, or electron microscopy. Alternatively, the effects oftreatments or compounds potentially affecting the expression of cellsurface proteins may be assayed by exposing the cells to eitherfluorescently labeled ligands of the proteins or antibodies to theproteins and then measuring the fluorescent emissions associated witheach cell on the plate. As another example, the effects of treatments orcompounds which potentially alter the pH or levels of various ionswithin cells may be assayed using various dyes which change in color atdetermined pH values or in the presence of particular ions. The use ofsuch dyes is well known in the art. For cells which have beentransformed or transfected with a genetic marker, such as theβ-galactosidase, alkaline phosphatase, or luciferase genes, the effectsof treatments or compounds may be assessed by assays for expression ofthat marker. In particular, the marker may be chosen so as to causespectrophotometrically assayable changes associated with its expression.

Particular screening applications of this invention relate to thetesting of pharmaceutical compounds in drug research. The reader isreferred generally to the standard textbook In Vitro Methods inPharmaceutical Research, Academic Press, 1997, and U.S. Pat. No.5,030,015. In certain aspects of this invention, the co-culture of theinvention is used to grow and differentiate hepatocytes to play the roleof test cells for standard drug screening and toxicity assays, as havebeen previously performed on hepatocyte cell lines or primaryhepatocytes in short-term culture. Assessment of the activity ofcandidate pharmaceutical compounds generally involves combining thehepatocytes with the candidate compound, determining any change in themorphology, marker phenotype, or metabolic activity of the cells that isattributable to the candidate compound (compared with untreated cells orcells treated with an inert compound), and then correlating the effectof the candidate compound with the observed change. The screening may bedone because the candidate compound is designed to have apharmacological effect on liver cells, or because a candidate compounddesigned to have effects elsewhere may have unintended hepatic sideeffects. Alternatively, libraries can be screened without anypredetermined expectations in hopes of identifying compounds withdesired effects.

In some embodiments, the co-culture of the invention is used to screenpharmaceutical compounds for potential cytotoxicity, such ashepatotoxicity (Castell et al., In: In Vitro Methods in PharmaceuticalResearch, Academic Press, 375-410, 1997. Cell Encapsulation Technologyand Therapeutics, Kuhtreiber et al. eds., Birkhauser, Boston, Mass.,1999). Cytotoxicity can be determined in the first instance by theeffect on cell viability, morphology, and leakage of enzymes into theculture medium. In certain embodiments, toxicity may be assessed byobservation of vital staining techniques, ELISA assays,immunohistochemistry, and the like or by analyzing the cellular contentof the culture, e.g., by total cell counts, and differential cell countsor by metabolic markers such as MTT and XTT.

In some embodiments, more detailed analysis is conducted to determinewhether the pharmaceutical compounds affect hepatic cell function (suchas gluconeogenesis, ureogenesis, and plasma protein synthesis) withoutcausing toxicity. Lactate dehydrogenase (LDH) is a good marker becausethe hepatic isoenzyme (type V) is stable in culture conditions, allowingreproducible measurements in culture supernatants after 12-24 hincubation. Leakage of enzymes such as mitochondrial glutamateoxaloacetate transaminase and glutamate pyruvate transaminase can alsobe used. Gomez-Lechon et al., Anal. Biochem., 236:296, 1996 describes amicroassay for measuring glycogen, which can be used to measure theeffect of pharmaceutical compounds on hepatocyte gluconeogenesis.

Other methods to evaluate hepatotoxicity include determination of thesynthesis and secretion of albumin, cholesterol, and lipoproteins;transport of conjugated bile acids and bilirubin; ureagenesis;cytochrome p450 levels and activities; glutathione levels; release ofalpha-glutathione s-transferase; ATP, ADP, and AMP metabolism;intracellular K+ and Ca2+ concentrations; the release of nuclear matrixproteins or oligonucleosomes; and induction of apoptosis (indicated bycell rounding, condensation of chromatin, and nuclear fragmentation).DNA synthesis can be measured as [³H]-thymidine or BrdU incorporation.Effects of a drug on DNA synthesis or structure can be determined bymeasuring DNA synthesis or repair. [³H]-thymidine or BrdU incorporation,especially at unscheduled times in the cell cycle, or above the levelrequired for cell replication, is consistent with a drug effect.Unwanted effects can also include unusual rates of sister chromatidexchange, determined by metaphase spread. The reader is referred toVickers in In Vitro Methods in Pharmaceutical Research, Academic Press,375-410, 1997 for further elaboration.

In certain embodiments, the invention includes a method for assessing anagent that alters hepatocyte biological activity, comprising contactinga hepatocyte present in the co-culture as described herein with anagent, and assaying for an alteration in hepatocyte biological activityrelative to a control hepatocyte not exposed to the agent, whereindetection of the alteration identifies the agent as altering hepatocytebiological activity. In certain embodiments, the hepatocyte biologicalactivity is selected from proliferation, survival, differentiation, andtoxicity, or combinations thereof. In certain embodiments, the assaycomprises measuring albumin output as a surrogate marker for proteinsynthesis; measuring urea generation as a surrogate marker for aminoacid metabolism function; and/or measuring cytochrome P450 activity as asurrogate marker for detoxification.

In certain embodiments, the invention provides a method for predictingthe effect of a test agent on epithelial cells, such as hepatocytes, ofa subject in vivo, comprising culturing epithelial cells obtained from asubject in the co-culture of the invention, exposing the epithelialcells to the test agent, and assaying for a pharmacological ortoxicological effect of the test agent on the epithelial cells relativeto control epithelial cells not treated with the test agent. In certainembodiments, a pharmacological or toxicological effect is selected fromproliferation, survival, differentiation, and toxicity, or combinationsthereof. In certain embodiments, the assay comprises quantifying thenumber of epithelial cells in the co-culture by using nuclearmorphologies to distinguish an epithelial cell from a feeder cell. Incertain embodiments, the assay is selected from measuring albuminsynthesis, urea secretion, and cytochrome p450 activity, or combinationsthereof.

In certain embodiments, the invention provides a method for assessingthe metabolism of a test agent by epithelial cells, preferablyhepatocytes, comprising exposing the co-culture of the invention to atest agent, and determining the effect of the epithelial cells on thetest agent. For example, the effect may be measured by detecting,identifying, and/or quantifying metabolites of the test agent.

In certain embodiments, the invention provides a method for predictingthe metabolism of a test agent by epithelial cells, preferablyhepatocytes, of a subject, comprising culturing epithelial cellsobtained from a subject in the co-culture of the invention, exposing theepithelial cells to the test agent, and determining the effect of theepithelial cells on the test agent. For example, the effect may bemeasured by detecting, identifying, and/or quantifying metabolites ofthe test agent.

Additional uses of the co-culture of the invention include, but are notlimited to, maintenance and expansion of epithelial cells, such ashepatocytes, for their use in transplantation or implantation in vivo;screening cytotoxic compounds, carcinogens, mutagens, growth/regulatoryfactors, pharmaceutical compounds, etc., in vitro; elucidating themechanism of liver diseases and infections; studying the mechanism bywhich drugs and/or growth factors operate; diagnosing and monitoringliver disease in a patient; gene therapy; and the production ofbiologically active products, to name but a few.

Additional further uses of the co-culture of the invention include, butare not limited to, its use in research e.g., to elucidate cellulargrowth mechanisms leading to the identification of novel targets forcancer therapies, to elucidate mechanisms involved in cell fatedetermination leading to new strategies for cellular reprogramming, andto generate genotype-specific cells for disease modeling, including thegeneration of new therapies customized to different genotypes. Suchcustomization can reduce adverse drug effects and help identifytherapies appropriate to the patient's genotype.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 Primary Human Hepatocytes Co-Culture Platform Design

During the initial testing of eight different donors of cryopreservedhuman hepatocytes, three were non-plateable, thus incompatible withphenotypic screening. While the remaining five donors all yieldedhepatocytes that adhered to rigid collagen in culture, one donor was tooyoung (0.1 years) to exhibit a full repertoire of mature hepatocytefunctions while another two donors had poor functions at baseline.Ultimately, donor GHA, who was a one-year-old Caucasian female with acause of death of dry drowning, was chosen. GHA hepatocytes attachedwell to rigid collagen and demonstrated good synthetic, detoxificationand metabolic functions.

TABLE 2 Donors of cryopreserved primary human hepatocytes Donor AgePlate-able? Function HU0845 47 yrs No N/A HU4122 19 yrs No N/A HU4088 2yrs No N/A HU4100 40 yrs Yes N/A KQG 38 yrs Yes Low SCT 38 yrs Yes LowRQO 0.1 yrs Yes Too young GHA (donor a) 1 yr Yes Good

Eight different donor lots were tested for suitability forhigh-throughput screening through examination of plate-ability andbaseline functions such as albumin secretion, urea production andcytochrome P450 activity.

To maintain primary human hepatocytes in culture, they wereco-cultivated with murine embryonic J2-3T3 fibroblasts, which have beenshown to transiently stabilize hepatocytes in vitro. This co-cultureeffect is mediated by heterotypic cell-cell contact between primaryhepatocytes and stromal cells as well as continuous stimulation withstromal-derived, short-range signaling molecules.

There exist multiple configurations of co-cultures of primaryhepatocytes and J2-3T3 fibroblasts, with varying degrees ofarchitectural organization. The simplest implementation consists of aco-planar distribution of randomly mixed hepatocytes and J2-3T3fibroblasts on a matrix of rigid collagen type I. More sophisticateddesigns comprise the application of semiconductor-driven microtechnologyto organize primary hepatocytes into in vitro colonies of empiricallyoptimized island sizes, subsequently surrounded by J2-3T3 fibroblasts;this particular configuration is termed micro-patterned co-culture(MPCC). All configurations of hepatocyte-J2 co-cultures were found tomaintain primary human hepatocyte functions in vitro for at least 9days, but none recapitulates their innate potential for substantialproliferation.

In general, increased architectural organization of cells in cultureleads to longer-term stabilization of hepatocyte functions, with MPCCbeing the most optimal configuration, enabling maintenance of hepatocytefunctions in vitro for 4-6 weeks. However, while the packing ofhepatocytes onto pockets of circular islands tightly surrounded byJ2-3T3 fibroblasts maximizes the homotypic cell-cell interactions thatenhance long-term hepatocyte survival and function, the confluentcell-cell contact may prevent expansion of any cell type whoseproliferation is normally contact-inhibited. Additionally, MPCCs aredifficult to miniaturize beyond 96-well platforms and in any case, suchprolonged periods of hepatocyte functions are neither necessary norpractical for most whole-cell screens.

The high-throughput liver platform was designed to assume a feeder layerco-culture configuration in order to provide both time and space forhepatocyte expansion. The platform contained a sparse population ofhepatocytes on top of a confluent layer of J2-3T3s within 384-wellplates (FIG. 1A). This design enabled fibroblast-mediated hepatocytestabilization for at least 9 days (FIG. 1B) without hepatocyte crowdingand was amenable to 384-well and smaller formats.

The number of fibroblasts per well was empirically optimized to 8,000cells/well in order to establish a confluent feeder layer withoutcontraction and aggregation of overcrowded J2-3T3 fibroblasts. Thenumber of hepatocytes per well was empirically optimized to 2,000cells/well, minimizing the number of hepatocytes in culture to allowroom for expansion while balancing the need for sufficient albuminoutput detectable via ELISA (FIG. 2).

Similarly, the amount of media used per well was empirically optimizedto balance opposing needs of oxygen transport and nutrient supply—toomuch media presented a transport barrier for gas diffusion, causingobservable steatosis in cultured hepatocytes; too little media causednutrient deprivation. There was an additional restriction that allfluids handled robotically must be dispensed in volumes that aremultiples of 10 μl; while the robots can be programmed to dispense insingle microliter gradients, only multiples of 10 μl offered sufficientaccuracy. All cells were robotically seeded at the lowest possible speedsetting in order to minimize physical stresses. During pilot testing, itwas observed that fibroblasts had difficulty remaining attached to plaintissue culture plastic in 384-well formats, thus a matrix coating ofCollagen type I was added at a concentration of 100 ug/mL. To assesscell fates in this platform, two separate high-throughput readouts weredeveloped: an image-based proliferation assay and three biochemicalfunctional assays.

Example 2 Co-Culture Proliferation Assay

Conventional approaches of co-cultivation of hepatocytes with J2-3T3fibroblasts allows long-term maintenance of primary human hepatocytes inculture but renders the measurement of hepatocyte proliferationchallenging. The co-existence of two different cell types in each wellrequires a proliferation assay that is specific to hepatocytes. However,all existing measurements of cellular proliferation and number, such asAlamar Blue, Cell Titer Glow and cell cycle stains including Ki67, BrdUand PNA, all reflect the proliferation state of the whole well, whichallows behavior of the more populous J2-3T3 fibroblasts to mask moderatehepatocyte expansions in culture. Therefore, a custom image-basedreadout was developed to specifically measure hepatocyte proliferationin the high-throughput liver platform.

Hepatocytes in culture can be distinguished from underlying J2-3T3fibroblasts via a variety of methods, including phase-contrastmicroscopy, staining for hepatocyte-specific markers, such as Albuminand CD44, and striking differences in nuclear morphology. Brightfieldimages, while easy to acquire, are difficult to quantify, particularlyin a high-throughput manner Immunofluorescent staining of particularantigens, while easy to measure in an automated fashion, are difficultto execute in 384-well and smaller formats. Therefore, an image-basedproliferation assay was developed that used nuclear morphology toquantify hepatocyte nuclei numbers in culture. When visualized withHoechst stain, hepatocyte nuclei (FIG. 3, left image) were more uniformin texture while fibroblast nuclei are punctate (FIG. 3, right image).The assay thus visualized all cell nuclei (FIG. 4A) in culture using asimple Hoechst stain (FIG. 4B), specifically identified hepatocytesbased on nuclear morphology (FIG. 4C) and provided a count of the numberof hepatocyte nuclei in culture (FIG. 4D).

It should be noted that primary hepatocytes have been known to exist inmulti-nucleated states and/or initiate cell cycle without completingcytokinesis. Thus this assay was not strictly a measure of hepatocyteexpansion. However, cellular proliferation cannot occur without DNAsynthesis. The image-based assay was designed to minimize the loss ofactive molecules to false negative errors during primary screening asthey cannot be recovered later on; On the other hand, false positivemolecules that induce DNA synthesis and/or multi-nucleation withoutcellular expansion can be filtered away during secondary screening, invitro or in vivo hit validation.

Cultures of hepatocytes and J2-3T3 fibroblasts were fixed using 4%paraformaldehyde (PFA) in black-walled, clear and flat-bottomed 384-wellplates (Corning). Fixed samples were then stained with Hoechst 33342. Ofnote is that the cell membrane was much more permeable to Hoechst 33342than Hoechst 33258; thus an additional permeabilization step using 0.1%Triton-X for 30 minutes was necessary when visualizing nuclei withHoechst 33258. Without membrane permeabilization, Hoechst 33258 led toheterogenous staining intensities, which lowered the accuracy ofsubsequent image analyses (FIG. 5). Images of fluorescently labelednuclei were acquired and digitized using a high-throughput screeningmicroscope (Molecular Devices DM) coupled to a barcode reader androbotic arm (Thermo) for automated plate loading (FIG. 6). Themicroscope was configured to self-focus, first using lasers to identifythe bottom of wells via differences in the refractive index of plasticand fluids, then using image-based focusing algorithms that scannedthrough a z stack of ˜200 μm in ˜50 μm steps in search of the plane withthe sharpest images.

In earlier implementations of this image assay, the distinction ofhepatocytes from fibroblasts was explored using z-position alone, butthe differences between the two layers of the co-culture was too minute(˜5 μm). The greater number of fibroblast nuclei (8,000 fibroblasts vs.2,000 hepatocytes) and their punctate nature ensured automated focusingon the fibroblast plane, further enhancing the morphological differencesbetween hepatocytes and fibroblasts to facilitate subsequent imageanalyses. Self-focus mechanisms did occasionally fail, buryingpopulations of blurred images among successful acquisitions, depressingthe accuracy of hepatocyte nuclei counts. To address this, subsequentanalyses pipelines were developed to flag the occurrence of thesenormally rare failures using blurry nuclear morphologies.

Accurate examination of nuclear morphology required image acquisition at20× magnification. Given the large volume of images required to cover100% of well area at this high magnification, 50% of the well area in acheckerboard fashion was sampled, imaging a total of 13 sites per well.To speed up image acquisition, laser-based focusing occurred once perplate and image-based focusing executed once per well.

Automated image analysis pipelines were developed to identify everynucleus in every Hoechst-stained image of the screening cultures, and tomeasure various characteristics (e.g. shape, size, intensity, proximity,texture) of each nucleus using the open-source CellProfiler software.

An important first step in image processing was illumination correction.Illumination varied, in some instances, by more than 1.5-fold across afield of view, despite the use of fiber optic light sources. This addedan unacceptable level of noise, and compromised the accuracy ofsubsequent analyses involving object intensity, including nucleiidentification and classification. Thus for the proliferation assay, theCellProfiler was configured to stack all acquired images from a singleexperiment to identify and normalize consistent discrepancies in thestaining intensities across the field of view.

Nuclei identification or segmentation was challenging when source imageswere crowded or, in this case, also contained overlapping objects. Theaccuracy of this step played a central role in determining the accuracyof the resulting nuclei counts. A variety of object identificationmodules offered within CellProfiler were tested, starting with a modularstrategy that first identified object edges based on intensity, thenseparated clumped objects based on their measurements such as shape orsize. This module offered a great degree of versatility, allowingcustomization of a number of parameters such as expected object size andintensity thresholds. While this module was configured to successfullyidentify crowded nuclei, an accurate segmentation of overlapping objectswas difficult. Therefore, a custom module of nuclei segmentation wasassembled.

The segmentation pipeline implemented in the proliferation assay firstused relative peaks in intensity to pinpoint positions of potentialnuclei, so that overlapping nuclei could be correctly identified asseparate objects. After the locations of nuclei were found, their edgescould then be outlined more accurately using Propagation algorithms.Test modules were implemented that compared several algorithms side byside in order and incorporated into the pipeline the most accuratesegmentation algorithm (FIG. 7).

For each identified nucleus, a large number of features were measured toconstruct a nuclear profile, including nuclear size, shape, intensity,proximity, and texture. This profile was subsequently used to trainmachine learning algorithms to automatically classify nuclei ashepatocytes or fibroblasts. During assay development, nuclear morphologywas observed to most effectively distinguish fibroblast nuclei fromhepatocyte nuclei as there were punctate sub-nuclear structures presentin fibroblasts, but absent in hepatocytes. Unfortunately, these punctatestructures, while numerous, were very small, thus occupying only aminute percentage of the nuclei area; consequently, their impact on themeasurements at the whole-nucleus level was too dilute for effectivemachine training. To address this, an additional segmentation module wasincluded that identified the punctate sub-nuclear structures andmeasured how many and what type (e.g. big or small, bright or dull) ofpunctuates were associated with each nucleus (FIG. 8).

The nuclear profiles generated by CellProfiler were inputted intoCellProfilerAnalyst178 for training of machine learning algorithms todistinguish and count hepatocyte nuclei. The training phase was manuallyinitiated by identifying a few hepatocytes and a few fibroblasts. Toavoid over-fitting the machine learning algorithm to a few particularsamples, the initial training sets were populated with ˜50 hepatocytesand ˜50 fibroblasts taken randomly from the general population withoutreferences to specific wells or plates. Using this initial training set,a machine learning algorithm was used to generate a preliminary set ofrules for nuclei classification, using the GentleBossting algorithmapplied to regression stumps. This rule set was used byCellProfilerAnalyst to classify a new batch of nuclei, outputting theresults for manual error correction. The corrections were then used torefine the rule set in an iterative process until an accuracy plateau isreached. Once finalized, the rule set was applied to the nuclearprofiles of every nucleus of every image in the experiment to classifyeach object as a hepatocyte or fibroblast before outputting a count ofeach nucleus type per well.

Initial training was conducted without info on sub-nuclear structuresand required approximately 1 day to complete a training set containing˜5000 manually classified objects with an accuracy plateau of ˜75% usinga total of 300 rules. With the assistance of punctate sub-nuclearstructures, this assay used only a few hours to generate a training setof ˜500 hundred objects with an accuracy plateau of at least 90%, often95%, using a total of 100 rules (FIG. 9).

Assay readiness for high-throughput screening is most often assessed viastatistical parameters such as z′-factor179, which reflects both assaysignal dynamic range and variation, and is mathematically defined:

$Z^{\prime} = {1 - \frac{\left( {{3\sigma_{c +}} + {3\sigma_{c -}}} \right)}{{\mu_{c +} - \mu_{c -}}}}$where “c+”=positive control, “c−”=negative control, “σ”=standarddeviation and “μ”=average. Assuming normal distribution, assays withpositive z′-factors can separate 99.8% of the negative and positivecontrol populations (i.e. the two populations, as defined by meansignal+/−3 standard deviations, do not overlap), essentially separatingsignal from noise.

Imaging of multiple 384-well plates containing untreatedhepatocyte-fibroblast co-cultures showed that the image-based readoutcan confidently (Z′>0) detect doublings in hepatocyte nuclei numberswith low variance (CV<20%) and good reproducibility. It should be noted,however, that the highly textured nature of fibroblast nuclei renderedtheir segmentation difficult, often leading to the breakup of a singlenucleus into multiple nuclei. Therefore, while the assay did reportnumbers of fibroblast nuclei as well as hepatocyte nuclei, it wasoptimized for accurate detection of hepatocyte nuclei.

In addition to quantifying hepatocyte nuclei in interphase, twoadditional analyses were developed to quantify the number of nuclei inthe process of mitosis. These analyses were built to detect nuclearmorphologies consistent with cells undergoing metaphase and anaphase(FIG. 10). While CellProfilerAnalyst was capable of simultaneouslyidentifying more than 2 morphologies, its classification accuracy becamesignificantly impaired with each additional category, thus separatetraining sets were generated for each morphology of interest (i.e.metaphase nuclei, anaphase nuclei and hepatocyte nuclei in interphase).

Cells undergoing metaphase have very distinctive nuclear morphologiesand were easily quantifiable using the image assay outlined earlier inthis section. Cells undergoing anaphase unfortunately assumed verysimilar morphologies to staining/camera artifacts, with just onedistinction: anaphase nuclei always appeared in closely positionedpairs. Therefore, minor adjustments were made to the measurements ofthese objects to include neighbor relationships.

Training of automated nuclei classification was also altered slightly toaccommodate the rare nature of these mitotic bodies. Instead ofpopulating the training set extensively with randomly selected objects,which would result in a severely imbalanced training set containingthousands of negative examples but only a few positive items, iterativeerror correction was focused on.

Validation of mitotic body detection was conducted manually throughvisual inspection of raw images due to the lack of a positive controlthat can induce proliferation of primary human hepatocytes. In general,program reported counts of mitotic bodies were in agreement withmanually obtained values.

In addition to the above image-based proliferation assay, thehigh-throughput liver platform was equipped with several functionalassays in order to probe whether hepatocytes in the platform retainedtheir liver identity. Due to the diverse repertoire of the 500+documented and yet unidentified biochemical functions of the liver,there does not exist a single all-inclusive, gold-standard assay formeasuring hepatocyte functions. Thus 3 major types of liver functionswere sampled: 1) ELISA-based quantification of albumin output as asurrogate marker for protein synthesis functions of the liver, 2)colorimetric assay quantifying urea generation as a surrogate marker foramino acid metabolism functions of the liver, 3) enzyme activity assaymeasuring cytochrome P450 activity as a surrogate marker fordetoxification functions of the liver.

For all 3 assays, parameters were optimized, such as reagent type,concentration and volume to develop them into biochemical assayscompatible with high-throughput screening, with Z′>0 and wide dynamicranges of detection (FIGS. 11A, 11B and 11C). Ultimately, for screeningpurposes, it is neither necessary nor practical to implement all 3assays, thus the ELISA-based albumin quantification was chosen as thefunctional assay for the human liver platform.

The most common form of the ELISA assay is a sandwich ELISA thatcaptures the antigen of interest in between 2 layers of antibodies. Thisassay is difficult to adapt to high-throughput screening due to the longprotocol, which limits through-put, and the extensive washes, which aredifficult to program robotically. Therefore, for the liver platform, acompetitive ELISA assay was employed, which reduced the length ofworkflow by approximately a third. A saturating amount of human albuminwas first coated onto the walls of adsorptive 384-well plates. Samplesupernatant was then introduced and competed with coated albumin forbinding to HRP-conjugated antibodies. The amount of bound antibodies wasthen quantified via a colorimetric substrate. Automation of the ELISAassay necessitated a few adjustments to the platform. Volume of mediaused to maintain cultures was increased to 30 ul/well in order to allowwithdrawal of 20 ul of sample without disturbing the cell layer. Anadhesive breathable membrane was added to the top of culture plates tominimize edge effects arising from fluid evaporation. The visualizationagent was changed from TMB to an ultra-sensitive luminescent substrateto increase sensitivity (FIG. 12). Ultimately, validation data showedthat this biochemical functional assay confidently (Z′>0) detecteddoublings in hepatocyte populations with low variance (CV<10%) and goodreproducibility.

Predictive high-throughput liver models are a critical tool for researchand development of novel therapeutics as well as for the study of liverbiology. Co-cultivation of hepatocytes with J2-3T3 fibroblastsrepresents a scale-able platform that can maintain primary humanhepatocytes in culture for at least 9 days, providing both time andspace for a wide range of cellular activities. The miniature feederlayer co-culture platform described herein for primary human hepatocytesand attendant assays are useful for probing multiple hepatocytephenotypes including cellular proliferation, cell death, proteinsynthesis functions, detoxification functions and amino acid metabolism.

Example 3 Design of a Primary Human Hepatocyte Co-Culture for ChemicalScreening

A high-throughput liver platform was developed to enable unbiasedchemical screening on primary human hepatocytes. The screen was designedto identify compounds that could induce functional proliferation and/ordifferentiation of the hepatocyte in order to generate renewable sourcesof functional human hepatocytes. The treatment of cells with smallmolecules has been shown to modulate a wide range of cellular processes.These processes include stem cell self-renewal and differentiation, andthe proliferation of normally quiescent mature adult cells, such aspancreatic β-cells and cardiomyocytes. Compounds can act through avariety of mechanisms to induce cell division, including activation ofdevelopmental signaling pathways such as Wnt or recruitment of GEFs tothe plasma membrane for RAS/MAPK pathway activation.

The accuracy and power of a high throughput screening (HTS) isdetermined largely by the quality of the biological platform and assayreadouts. Thus, a robust screening platform was developed for primaryhuman hepatocytes.

In order to avoid species-specific differences and cell line mutations,the screen was conducted with human primary hepatocytes. Traditionally,chemical screening on such cells has been hindered by their availabilityin large quantities as well as their rapid loss of viability andphenotype in vitro. Recent advances in cryopreservation technologiesallowed enough primary human cells to be stored for screening, and tomaintain these cells, the hepatocytes were co-cultivated withnon-parenchymal cells. Co-cultures of primary human hepatocytes withmurine embryonic J2-3T3 fibroblasts were recently shown to maintainnormal hepatocyte phenotype for weeks. These in vitro platformsconsisted of hepatocytes surrounded by a co-planar population offibroblasts. While capable of stabilizing hepatocyte functions in vitro,such platforms may limit normal hepatocyte expansion due to contactinhibition. Thus, to provide surface area for hepatocyte expansion, asparse population of hepatocytes was co-cultivated on top of a confluentfeeder layer of J2-3T3 fibroblasts within 384-well plates. Thisscreening platform stabilized hepatocyte phenotypic functions in vitroand was compatible with two separate high-throughput readouts developedfor this screen.

The primary readout detected hepatocyte proliferation via automatedhigh-content imaging. This assay quantified hepatocyte nuclei numbers,using nuclear morphologies to separate the hepatocyte and fibroblastsub-populations that co-exist within the screening platform. Whenvisualized with Hoechst stain, hepatocyte nuclei were smaller and moreuniform in texture, while fibroblast nuclei are larger and punctated(FIG. 13). Leveraging this distinction, automated image analyses wasdeveloped that utilized machine learning algorithms to classify nucleitypes and tabulate hepatocyte nuclei numbers. Assay validation datashowed that this image-based readout can confidently (z′>0) detectdoublings in hepatocyte nuclei numbers with low variance (CV<20%) andgood reproducibility. In addition to quantifying hepatocyte nuclei thathave completed mitosis, the number of nuclei in the process of mitosiswere also found and quantified. Two additional analysis pipelines werebuilt to detect nuclear morphologies consistent with cells undergoingmetaphase and anaphase.

In order to evaluate the phenotype of treated cells, a secondary readoutwas included to quantify hepatocyte functions via competitive ELISA.This biochemical assay measured the level of secreted albumin as amarker for protein synthesis functions of the cultured hepatocytes (FIG.13).

Example 4 Design of Image-Based Proliferation Assay Workflow

To detect hepatocyte proliferation, a proliferation readout that canseparate the hepatocyte and fibroblast sub-populations that co-existwithin the co-culture was developed (FIG. 4A). The proliferation readoutdetected hepatocyte proliferation via a customized, automated,high-content imaging protocol. In brief, the assay quantified the numberof hepatocyte nuclei, using nuclear morphologies to distinguish thehepatocyte and fibroblast sub-populations that co-exist within thescreening platform. When visualized with Hoechst stain, hepatocytenuclei were more uniform in texture while fibroblast nuclei werepunctate (FIG. 4B). An automated image analysis pipeline was developedthat identified every nucleus in every Hoechst-stained image of thescreening cultures, and to measured various characteristics (e.g. shape,size, intensity, proximity, and texture) of each nucleus using theopen-source CellProfiler software. Manual classification of randomlypresented nuclei and error correction of machine-classified nuclei wasincorporated into the nuclei classifier training (FIG. 4C). Thesecharacteristics morphologies were used to train machine learningalgorithms to identify and count the number of hepatocyte nuclei in eachimage (FIG. 4D).

Example 5 Production of Co-Culture Platform and HST Screening

384-well screening plates (Corning) were incubated with a solution oftype-I collagen in water (100 mg/ml, BD Biosciences) for 1 hour at 37°C. in order to allow for more secure cell attachment. A feeder layer ofJ2-3T3 fibroblasts were robotically plated onto the collagen at adensity of 8,000 cells/well (designated as day −2), and allowed to reachconfluency over 48 hours, when their growth became contact inhibited.Hydrocortisone in the culture medium curbed further fibroblast expansionto prevent overgrowth of the feeder layer.

Primary human hepatocytes were plated onto the fibroblasts on day 0 at adensity of 2,000 cells/well and maintained under standard cultureconditions with daily replacement of hepatocyte medium for 7 days.Primary human hepatocytes were purchased in cryopreserved suspensionfrom Celsis In vitro Technologies (donor a) and Invitrogen (donor b),and pelleted by centrifugation at 50 g for 10 minutes. The supernatantwas discarded before re-suspension of cells in hepatocyte culturemedium, which consisted of DMEM with high glucose, 10% (vol/vol) FBS,15.6 ug/mL insulin, 16 ng/ml glucagon, 7.5 ug/ml hydrocortisone and 1%(vol/vol) penicillin/streptomycin. A library of 12,480 compounds (FIG.14) was added on day 7 at a final concentration of ˜15 uM, and allowedto incubate for 48 hours. On day 9, culture supernatants were collectedfor automated ELISA analysis, and cells were fixed in 4% PFA for imaginganalysis (FIG. 15). Hepatocyte functions were determined via competitiveELISA (MP Biomedicals) using horseradish peroxidase detection andchemiluminescent luminol (Pierce) as a substrate. The cell-free counterassay involved ELISAs on fresh media incubated for 48 hrs with compoundsof interest. Hepatocyte proliferation was assessed through customized,automated high-content imaging protocol. Fixed cells were permeabilizedwith 0.1% Triton-X, nuclei visualized with Hoechst stain (Invitrogen)and robotically imaged (Thermo, Molecular Devices) at 25 dispersed sitesper well. Images were digitized and analyzed using CellProfiler andCellProfiler Analyst (Broad Institute).

Example 6 Methods and Materials

Luminex Analysis:

Cells were lysed using RLT buffer (Qiagen) or Trizol (Invitrogen) andpurified using the Mini-RNeasy kit (Qiagen). Gene expression wasdetermined using Luminex analysis. Briefly, total RNA was immobilized ona Qiagen turbo capture 384-well plate, and reverse-transcribed usingoligo dT priming. A biotinylated FlexMap tag sequence unique to eachgene of interest and a phosphorylated downstream probe were then addedto resulting cDNAs to generate biotinylated FlexMap-tagged amplicons.Universal PCR was then performed for 35 cycles using a biotinylated T7forward primer and T3 reverse primer in buffer with dNTPs and Taqpolymerase. FlexMap microsphere beads conjugated with antitagoligonucleotides were then added and allowed to hybridize. Ampliconswere captured by streptavidin-phyoerythrin, and 100 events per bead wereanalyzed for internal bead color and phyoerythrin reporter fluorescenceon a Luminex FlexMap 3D analyzer. Data for replicate loadings, expressedin mean fluorescent intensity of at least 100 beads per sample, werescaled to the human transferrin gene and row-normalized for heat maprepresentation using GeneE open software (Broad Institute).

Biochemical Assays:

Culture media were collected and frozen at −20° C. until analysis.Albumin content was measured through sandwich ELISA assays (MPBiomedicals, Fitzgerald, Bethyl Laboratories) using horseradishperoxidase detection and 3,3′,5,5′-tetramethylbenzidine (TMB, FitzgeraldIndustries) as a substrate. Urea concentration was determinedcolorimetrically using diacetylmonoxime with acid and heat (StanbioLabs). To quantify CYP450 activity, at 48 hours after small moleculeexposure, cultures were incubated with substrates (coumarin from Sigmafor CYP2A6, luciferin-IPA from Promega for CYP3A4) for 4 hours at 37° C.Incubation medium was collected and metabolite concentration quantifiedvia luminescence, or fluorescence after hydrolization of potentialmetabolite conjugates by β-glucuronidase/arylsulfatase (Roche, Ind.).

Cell Counting:

J2-3T3 fibroblasts were covalently labeled with Cell Tracker CM-DiI(Invitrogen) before initiation of co-culture. For FACS analysis, cellswere treated with collagenase, then accutase, and suspended in PBS-0.2%FBS. Cell suspensions were supplemented with 50,000 fluorescent countingbeads (CountBright, Invitrogen) per sample. Data were acquired with a4-color flow cytometer (FACSCalibur, BD Biosciences) and analyzed withCellQuest (BD Biosciences). For Cellometer analysis, cells weretrypsinized and placed on cell counting chambers (Nexcelom) forautomated cell counting.

Hepatocyte Medium Composition:

1×DMEM

10% fetal bovine serum (FBS)

15.6 ug/ml insulin

7.5 μg/ml hydrocortisone

16 ng/ml glucagon

1% penicillin-streptomycin

Fibroblast Medium Composition:

1×DMEM

10% bovine serum (BS)

1% penicillin-streptomycin

J2-3T3 Culture Conditions.

Passage 2 J2-3T3 fibroblasts were obtained from Howard Green (Harvard)and kept in liquid nitrogen until use. Cells were maintained understandard tissue culture conditions, in DMEM media containing 10% BS and1% Penicillin-streptomycin. Fibroblasts were grown in T-150 tissueculture flasks and passaged 1:10 using 0.25% Trypsin-EDTA when cellsreached confluency. Experiments used J2-3T3s ranging in passage numbersfrom P9 to P12.

Automated Cell Seeding.

Cells suspensions were diluted to the desired densities and kept insuspension using a magnetic stir bar. Thermo Combi robot was used todispense cells into 384-well formats using speed setting low andstandard cassette.

Biochemical Assays.

Urea concentration was quantified using a colorimetric assay thatemploys diacetylmonoxime with acid and heat (Stanbio Labs). Albumincontent was measured using ELISA assays (MP Biomedicals) withhorseradish peroxidase detection and TMB (Fitzgerald Industries)substrate.

Cytochrome-P450 Induction.

7-benzyloxy-4-trifluoromethylcoumarin (BFC, BDGentest) was added tocultures at 50 uM and incubated for 1 hr at 37° C. in phenol-red freemedia. Many different CYP450 isoforms process BFC into its fluorescentproduct of 7-hydroxy-4-trifluoromethylcoumarin (7-HFC), which is thenquantified fluorometrically.

Automated Plate Washing.

Washing for plates containing cells were done manually to prevent cellloss. Plate washing for ELISA was performed on the BioTek ELx-405 HT,using the following optimized settings:

Prime: Prime_200 using DI water

Wash: Named program HEPELISA

Method

-   -   Number of cycles=02    -   Wash Format=Plate    -   Soak/Shake=Yes    -   Soak Duration=010 sec    -   Shake before soak=yes    -   Shake Duration=005 sec    -   Shake Intensity=4 (18 cycles/sec)    -   Prime after soak=No

Disp

-   -   Dispense volume=100 ul/well    -   Dispense flow rate=05    -   Dispense height=120 (15.240 mm)    -   Horizontal X disp pos=25 (1.143 mm)    -   Horizontal Y disp pos=20 (0.914 mm)    -   Bottom wash 1st=no    -   Prime before start=no

Aspir

-   -   Aspir. Height=020 (2.540 mm)    -   Horiz. X Asp. Pos=00    -   Horiz Y asp pos=00    -   Asp rate=05 (6.4 mm/sec)    -   Asp delay=0000 msec    -   Cross-wise aspir=yes    -   Cross-wise on=all    -   Cross-wise height=020 (2.540 mm)    -   Cross-wise X horiz. Pos=00    -   Cross-wise Y horiz. Pos=00    -   Final asp=Yes    -   Final asp. Delay=0000 msec

Automated Plate Reading.

Perkin Elmer Envision 2102 Multilabel Reader was used to quantify ELISAsignal. Program named ShanMeghan Chemillum and contains integrationduration of 0.1 sec, luminescence mirror, luminescence 700 emissionfilter and measurement height of 6.5 mm.

OTHER EMBODIMENTS

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A co-culture for high throughput analysis ofprimary hepatocytes, comprising: a layer of feeder cells disposedwithout aggregation in a well of a multi-well plate comprising at least96 wells; a layer of primary hepatocytes overlaid on the feeder cellswherein the hepatocytes are not contact inhibited and are at a densitythat allows for expansion of the hepatocytes in the co-culture for atleast 7 days prior to a high throughput analysis; wherein the bottomsurface of the well in the multi-well plate is coated with a celladhesion substrate selected from the group consisting of collagen,fibronectin, vitronectin, laminin, entactin, Arg-Gly-Asp (RGD) peptide,Tyr-Ile-Gly-Ser-Arg (YIGSR) peptide, glycosaminoglycans (GAGS),hyaluronic acid (HA), integrins, intercellular adhesion molecules(ICAMs), selectins, cadherin, cell-surface protein-specific antibodies,and a combination thereof; and wherein the feeder cells are disposed ina single confluent layer on the cell adhesion substrate; and culturemedium in the well of the multi-well plate in an amount sufficient tosupport hepatocyte expansion and maintain at least one biologicalactivity of the hepatocytes for assessment in the high throughputanalysis.
 2. The co-culture of claim 1, wherein the multi-well platecomprises at least 384 wells.
 3. The co-culture of claim 1, wherein thehepatocytes and feeder cells are plated at a ratio of 1:4.
 4. Theco-culture of claim 1, wherein the hepatocyte biological activity isselected from the group consisting of albumin secretion, liver-specificprotein synthesis, bile production, detoxification of compounds, energymetabolism, and cholesterol metabolism.
 5. The co-culture of claim 1,wherein the feeder cells and hepatocytes are of different species. 6.The co-culture of claim 1, wherein the feeder cells comprise one or moretypes of non-parenchymal cells.
 7. The co-culture of claim 6, whereinthe non-parenchymal cells are selected from the group consisting offibroblast or fibroblast-derived cells and hepatic non-parenchymalcells.
 8. The co-culture of claim 7, wherein the hepatic non-parenchymalcells are selected from the group consisting of Kupffer cells, Itocells, endothelial cells, stellate cells, cholangiocytes and hepaticnatural killer cells.
 9. The co-culture of claim 1, wherein the feedercells express a protein selected from the group consisting of Delta-likehomolog 1; C-fos-induced growth factor; Ceruloplasmin; Decorin;Interferon regulatory factor 1; 204 interferon-activatable protein;Splicing factor, arginine/serine-rich 3; JKTBP; Autoantigen La; Highmobility group box 1; Esk kinase; dihydrofolate reductase gene: 3′ end;Pm1 protein and Rac GTPase-activating protein
 1. 10. The co-culture ofclaim 1, wherein the culture medium comprises hydrocortisone.
 11. Theco-culture of claim 1, wherein the primary hepatocytes are overlaid onthe feeder cells in an amount of about 5×10³ cells/well or fewer in amulti-well plate having 384 wells.
 12. The co-culture of claim 1,wherein the hepatocytes are stable in the co-culture for at least 9 dayswithout hepatocyte crowding.
 13. A co-culture platform for highthroughput analysis of primary hepatocytes, comprising: a layer offeeder cells disposed without aggregation in a well of a multi-wellplate comprising at least 96 wells; a layer of primary hepatocytesoverlaid on the feeder cells wherein the hepatocytes are not contactinhibited and are at a density that allows for expansion of thehepatocytes in the co-culture for at least 7 days prior to a highthroughput analysis; wherein the bottom surface of the well in themulti-well plate is coated with a cell adhesion substrate selected fromthe group consisting of collagen, fibronectin, vitronectin, laminin,entactin, Arg-Gly-Asp (RGD) peptide, Tyr-Ile-Gly-Ser-Arg (YIGSR)peptide, glycosaminoglycans (GAGS), hyaluronic acid (HA), integrins,intercellular adhesion molecules (ICAMs), selectins, cadherin,cell-surface protein-specific antibodies, and a combination thereof;wherein the feeder cells express a protein selected from the groupconsisting of Delta-like homolog 1, C-fos-induced growth factor,Ceruloplasmin, Decorin, Interferon regulatory factor 1, 204interferon-activatable protein, Splicing factor, arginine/serine-rich 3,JKTBP, Autoantigen La, High mobility group box 1, Esk kinase,dihydrofolate reductase gene: 3′ end, Pml protein and RacGTPase-activating protein 1; and wherein the feeder cells are disposedin a single confluent layer on the cell adhesion substrate; and culturemedium in the well of the multi-well plate in an amount sufficient tosupport hepatocyte expansion and maintain at least one biologicalactivity of the hepatocytes for assessment in the high throughputanalysis.
 14. The co-culture of claim 13, wherein the hepatocytebiological activity is selected from the group consisting of albuminsecretion, liver-specific protein synthesis, bile production,detoxification of compounds, energy metabolism, and cholesterolmetabolism.
 15. The co-culture of claim 13, wherein the feeder cells andhepatocytes are of different species.
 16. The co-culture of claim 13,wherein the feeder cells comprise one or more types of non-parenchymalcells.
 17. The co-culture of claim 16, wherein the non-parenchymal cellsare selected from the group consisting of fibroblast orfibroblast-derived cells and hepatic non-parenchymal cells.
 18. Theco-culture of claim 17, wherein the hepatic non-parenchymal cells areselected from the group consisting of Kupffer cells, Ito cells,endothelial cells, stellate cells, cholangiocytes and hepatic naturalkiller cells.
 19. The co-culture of claim 13, wherein the primaryhepatocytes are overlaid on the feeder cells in an amount of about 5×10³cells/well or fewer in a multi-well plate having 384 wells.
 20. Theco-culture of claim 13, wherein the hepatocytes are overlaid onto thefeeder cells at a ratio of 1:4 or less.
 21. The co-culture of claim 13,wherein the multi-well plate comprises at least 384 wells.
 22. Theco-culture of claim 13, wherein the hepatocytes are stable in theco-culture for at least 9 days without hepatocyte crowding.